The present invention relates to a biosensor that carries out high-speed, highly-accurate, simple determination of a target object in a sample.
A biosensor has been proposed to determine a specific component in a sample by a simple procedure without any dilution or stirring the sample solution (Japanese Laid-Open Patent Publication No. 2-062952).
In this biosensor, an electrode system including a measurement electrode or a working electrode, a counter electrode, and a reference electrode is formed on an insulating base plate, for example, by screen printing. An enzyme reaction layer including a hydrophilic polymer, an oxidation-reduction enzyme, and an electron mediator is then formed on the electrode system. A buffer may be added to this enzyme reaction layer according to the requirements.
When a sample solution containing a substrate is added dropwise onto the enzyme reaction layer in the biosensor thus constructed, the enzyme reaction layer is dissolved to cause a reaction of the enzyme with the substrate, which results in reduction of the electron mediator. After completion of the enzyme reaction, the reduced electron mediator is oxidized electrochemically, and the concentration of the substrate included in the sample solution is calculated from the observed value of oxidation current.
In principle, the biosensor is applicable to measurement of diverse substances by selecting an appropriate enzyme that reacts with a target substance of measurement as the substrate. For example, when glucose oxidase is used as the oxidation-reduction enzyme, the biosensor is constructed to measure the concentration of glucose in blood. This is widely used as a glucose sensor. Application of cholesterol oxidase gives a biosensor that measures cholesterol in serum.
The value of serum cholesterol generally used for the index of diagnosis is the sum of the concentrations of cholesterol and cholesterol ester. The cholesterol ester is, however, not the substrate of the oxidation reaction with cholesterol oxidase. In order to measure the value of serum cholesterol as the index of diagnosis, an additional process is thus required to change the cholesterol ester to cholesterol. Cholesterol esterase is used as the enzyme that catalyzes this process.
The biosensor including cholesterol esterase and cholesterol oxidase in its enzyme reaction layer is used to measure the total concentration of cholesterol in serum.
The measurement of cholesterol is affected by cholesterol that is present in the cell membrane. The coexistence of a surface active agent with cholesterol esterase in the reaction reagent layer is preferable to enhance the reactivity. The surface active agent destroys the cell membrane in many cases, and there is a possibility that the substances inside the cell directly or indirectly affect the enzyme reaction or the electrode reaction. From this point of view, it is preferable that the enzyme reaction and the subsequent electrode reaction proceed in plasma or serum in the cholesterol sensor. In biosensors other than the cholesterol sensor, the presence of hemocytes in blood may also affect the response. It is accordingly ideal that the enzyme reaction and the electrode reaction proceed in a solution free of hemocytes.
Centrifugation is a known method to separate plasma or serum from whole blood. The centrifugation method, however, takes a rather long time and requires complicated operations.
U.S. Pat. No. 3,607,092 discloses a membrane used for testing blood. This membrane has a thin film layer that has permeability to liquids but impermeability to solids like hemocytes and giant molecules like protein. Namely this thin film functions to filter out the hemocytes. However, since the solid component is accumulated on the thin film with the passage of blood, a large area of the thin film layer is required to obtain filtrate of a certain amount sufficient for the reaction of the biosensor. The above-mentioned thin film is thus not sufficient.
U.S. Pat. No. 4,477,575 discloses an apparatus for and a method of separating serum from whole blood passing through a glass fiber filter. The method of separating serum from whole blood with a fiber or porous filter is applicable to the biosensor. This method, however, does not make the hemocytes kept in the filter but simply slows down the flow of hemocytes for separation of plasma. In the case of application of this method to the biosensor, a certain quantity of filtered plasma or serum sufficient for the reaction in the biosensor should be obtained, before the hemocytes are flown out of the filter. For this purpose, a specific setting that satisfies this condition should be applied for the length of the filter in the direction of blood flow.
The filter satisfying this condition is disposed between one portion of the biosensor with the electrode system and the reaction reagent system and another portion of the biosensor for supplying blood as a sample to construct the biosensor having the ability of filtering the hemocytes. FIG. 9 shows a biosensor of such construction. FIG. 9 is a decomposed perspective view of the biosensor without the reaction reagent layer.
In the example of FIG. 9, silver paste is printed on an insulating base plate 101 composed of polyethylene terephthalate by screen printing to form leads 102 and 103 and the base of an electrode system. Conductive carbon paste including a resin binder is printed on the base plate 101 to form the electrode system including a working electrode 104 and a counter electrode 105, while insulting paste is printed to form an insulating layer 106. The working electrode 104 is connected to the lead 102, and the counter electrode 105 to the lead 103. The insulating layer 106 makes the exposed area of the working electrode 104 and the counter electrode 105 constant, and partly covers the leads.
The process arranges the insulating base plate 101 with the electrode system, a cover 108 with an air vent 109, a spacer 107, and a filter 111 having the ability of filtering hemocytes at the positional relationship shown by the one-dot chain line and joins together to assemble a biosensor. The filter 111 is cut to fit a sample solution supply pathway, which is defined by a slit 110 of the spacer 107 between the cover 108 and the insulating base plate 101. Numeral 113a represents a portion at which the filter 111 is in contact with the insulating base plate. The filter 111 is disposed between the electrode system and a sample supply unit 112 on the base plate without covering over the electrode system including the working electrode 104 and the counter electrode 105 in the sample solution supply pathway.
In the biosensor having the above construction, blood added dropwise onto the sample supply unit 112 soaks into an end of the filter 111 close to the sample supply unit. In the filter, the permeation rate of hemocytes is less than the permeation rate of plasma as the liquid component, and the plasma accordingly soaks out of the end of the filter close to the electrode system. The soak-out plasma dissolves reaction reagents, which include enzymes and are carried at a specific position covering over the electrode system or on the rear face of the cover immediately above the specific position, and fills the whole sample solution supply pathway from the vicinity of the electrode system to the air vent 109. When the whole sample solution supply pathway is filled with the liquid, the flow of the liquid in the filter 111 stops, so that the hemocytes do not reach the end of the filter close to the electrode system but are retained at the current position.
Through the filtration of hemocytes, the reaction reagent layer dissolved in plasma chemically reacts with a target component included in the plasma, cholesterol in the case of the cholesterol sensor. After elapse of a preset time, the value of electric current is measured by means of the electrode reaction. This determines the component in the plasma.
In this prior art biosensor, part of the blood added dropwise to the sample supply unit 112 is not absorbed through the end of the filter 111 close to the sample supply unit. But the blood including hemocytes is transferred through the gap between the sample solution supply pathway and the filter 111 to reach the reaction reagent layer. This causes the hemocytes or some component in the hemocytes to react with the reaction reagent and give a significant error to the measurement.
Bonding the filter 111 to the sample solution supply pathway via an adhesive may prevent the transfer of blood through the gap between the filter 111 and the sample solution supply pathway.
The adhesive may, however, affect the blood components. This method also requires application of the adhesive on either the surface of the filter 111 or the sample solution supply pathway, which results in the complicated manufacturing process.
The object of the present invention is thus to solve the drawbacks discussed above by improving a biosensor with a filter that is capable of filtering a solid component like hemocytes.
More specifically the object of the present invention is to provide a biosensor that has stable response by allowing a sample added to the sensor to soak into a filter and making only a sample solution transmitted through the filter reach a reaction reagent layer and an electrode system.
A biosensor in accordance with the present invention includes: an insulating base plate, an electrode system that is provided on the base plate and has at least a working electrode and a counter electrode, a cover member that is combined with the base plate to define a sample solution supply pathway for leading a sample solution from a sample supply unit to the electrode system, a reaction reagent system including at least an oxidation-reduction enzyme and an electron mediator, and a filter disposed between the electrode system and the sample supply unit in the sample solution supply pathway, the biosensor having a space that encircles surface of the filter in an area from one end of the filter close to the sample supply unit to the other end of the filter close to the electrode system.
In one preferred mode of the present invention, the sample supply unit is provided on the base plate, and the sample solution supply pathway is formed along the base plate and the cover member.
In this mode, it is desirable that the space surrounding the surface of the filter has a width of not less than 0.5 mm. The width of the space less than 0.5 mm may cause blood transmitted through a gap between the base plate and/or the cover member forming the sample solution supply pathway and the filter to reach the area of the space by means of capillarity. The preferable width of the space ranges from 0.5 mm to 5.0 mm. The width over 5.0 mm may undesirably lead to deformation of the filter under vibrations applied to the sensor. More specifically, the preferable width is 1.0 mm to 3.0 mm.
In another preferred mode of the present invention, the sample supply unit is provided on the cover member, and the sample solution supply pathway is disposed in a direction of gravity from the sample supply unit. In this mode, it is preferable that the width of the space surrounding the surface of the filter is not less than 100 xcexcm and is smaller than the thickness of the filter.
The filter used here is a porous body having spaces connecting with one another in a three-dimensional manner, and the porous body moves blood from the sample supply unit toward the sample solution supply pathway by capillarity while functions to filter hemocytes based on a difference between flow resistances of plasma and the hemocytes. A non-woven fabric preferably composed of a hydrophilic fiber, such as fiber glass, cellulose, or pulp, filter paper, or another porous body may be applied for the filter.
The arrangement of the present invention is preferably applied for a cholesterol sensor in which the oxidation-reduction enzyme is cholesterol oxidase.
In the cholesterol sensor, it is preferable that the reaction reagent system includes an enzyme having an ability of hydrolyzing cholesterol ester. It is also preferable that the enzyme having the ability of hydrolyzing cholesterol ester is cholesterol esterase and that the reaction reagent system includes a surface active agent.
It is desirable that part or all of the cover member and the insulating base plate are transparent.